The surface charge of objects in the nanometre and micron range is a key factor in their stability and general reactivity. Surface charge and changes in surface charge can also be used to detect and quantify the presence of specific targets in a sample. The ability to accurately measure the surface charge of objects is therefore important for a range of users from industrial (e.g. paint, ink and coating manufacture) through to pharmaceutical (e.g. vaccine QA) and scientific research (e.g. developing nanomedicines and specific molecular diagnostic tests).
Current practice for measuring the surface charge of particles includes Dynamic Light Scattering (DLS) (Phase Analysis Light Scattering-Quasi Electric Light Scattering). Dynamic Light Scattering is used to measure particle and molecule size. This technique measures the diffusion of particles moving under Brownian motion, and converts this to size and a size distribution using the Stokes-Einstein relationship.
In conjunction with DLS Laser Doppler Micro-electrophoresis is used to measure the zeta potential or surface charge of particles. In this method an electric field is applied to a solution of molecules or a dispersion of particles, which then move with a velocity related to their zeta potential. This velocity is measured using a laser interferometric technique (Phase Analysis Light Scattering or PALS). This enables the calculation of electrophoretic mobility, and from this the zeta potential and zeta potential distribution.
However, the present inventors have appreciated that there are limitations to this approach. Due to the nature of the analysis individual particle sizes and zeta potentials are not measured, this is an “ensemble” technique. Separating similarly sized populations based on surface charge is therefore not possible; neither is identification of a subtle shift in surface charge of a group of particles, which could be used as a diagnostic measurement. The present inventors have appreciated that this limits the range of applications to which the technique may be applied.
A further limitation of DLS analysis, as appreciated by the present inventors, is that the average size distribution of a mixed population of particles is artificially skewed towards the larger particles, which scatter so much light that they can obscure smaller particles.
Another method used for the measurement of particle surface charge is Nanoparticle Tracking Analysis or NTA. NTA, like DLS, measures the Brownian motion of nanoparticles in 2 dimensions whose speed of motion, or diffusion coefficient, Dt, is related to particle size through the Stokes-Einstein equation. In NTA this motion is analysed by video; individual particle positional changes are tracked in two dimensions from which the particle diffusion is determined. Knowing Dt, the particle hydrodynamic diameter can be then determined on a particle by particle basis. By applying a bias voltage to the sample, NTA can also measure electrophoretic velocity and derive a zeta potential.
The present inventors have appreciated that limitations of NTA include the fact that a relatively small number of particles are analysed, and that it is possible to measure the same particle several times. Further, complex algorithms are applied to calculate the particle size and charge, and if the input settings are incorrect or not known the answer can be significantly erroneous. Additionally, compensation factors also need to be applied to balance both electro-osmotic flow and convection due to laser heating effects.
Another method used specifically for the measurement of particle surface charge is through the use of particle translation rate or duration as measured through Resistive Pulse Sensing.
Various techniques have been described to measure the relative electrophoretic velocity of populations of particles by adjusting either the voltage or the pressure applied across a nanopore and observing the change in either particle translocation rate or duration (typically FWHM) of the resistive pulses.
Vogel (Vogel, R., et al., “A Variable Pressure Method for Characterizing Nanoparticle Surface Charge Using Pore Sensors”. Analytical Chemistry, 2012. 84(7): p. 3125-3131.) describes one such application where sample particles are placed on both sides of the nanopore, and the pressure applied across a nanopore is adjusted until the flow of particles reverses direction. The applied pressure required to reach this “inversion point” is used as a measure of electrophoretic mobility. At the inversion point, the time it takes for particles to translocate the pore tends towards infinity, so translocation duration can also be used as an indicator.
As appreciated by the present inventors, the main limitations of these methods are that they are ensemble methods (not particle by particle), and that they are time-consuming as they require the adjusting of many parameters and many measurements. Additionally, with the pressure-sweep method in particular, the, duration of the measurement and the reversal of the fluid flow greatly increase the chance of a particle partially blocking the sensing zone.
Another method for the measurement of surface particle charge is described by Kozak (Kozak, D., et al., “Simultaneous size and ζ-potential measurements of individual nanoparticles in dispersion using size-tunable pore sensors”. ACS nano, 2012. 6(8): 6990-6997.). This method provides single particle charge measurements through analysing resistive pulse data. Kozak et al. describes a method for modelling a conical nanopore and calculating all of the forces on a particle passing through the nanopore. This method produces particle by particle surface charge data that can distinguish similarly sized particles based on surface charge, and gives consistent Zeta potential results for different bias voltages.
The present inventors have appreciated that this method has the disadvantage that the geometry and zeta potential of the sensing pore must be precisely known, must be invariant during analysis, and able to be modelled. In the example described by Kozak et al. the geometry of the pore is assumed to be perfectly conical, and calculations are based on that assumption. The present inventors have appreciated that in practice pores are typically not conical, and in many cases are not even rotationally symmetrical. In application the method described by Kozak et al. requires that the precise geometry and dimensions of a sensing pore are measured and modelled and that the pore charge is accurately known. This is commercially impractical. Simple application of the conical model described by Kozak et al. as an approximation is also impractical as this requires that the actual size in nanometres of the pore entry and exit must be known along with the actual pore charge. The present inventors have therefore appreciated that in practice the ability of the model to correctly predict fluid flow under different pressures is very limited.
A final method, described by Arjmandi et al, (Arjmandi et al., “Measuring the electric Charge and Zeta Potential of Nanometer-Sized Objects Using Pyramidal-Shaped Nanopores”. Analytical Chemistry, 2012. 84:8490-8496) is based on measuring the duration of the translocation of particles through a nanopore as a function of applied voltage.
In addition to the disadvantages described above, further disadvantages of existing methods, as appreciated by the present inventors, include:
1. Methods based purely on electrophoresis may be impractical. For example, samples of particles with a wide spectrum of zeta-potentials, potentially reaching from positive to negative values, may require the application of an external pressure in order to capture the whole spectrum of particle zeta-potentials. In addition, when measuring the charge/zeta-potential of very dilute particle suspensions, methods purely based on electrophoresis become impractical due to low count rates. Further, when measuring particle suspensions with a large proportion of nearly neutral particles in the absence of any applied pressure, the majority of the neutral particles may not translocate the pore, and hence not be measured, skewing the results.2. Changes in zeta potentials of the pore material are often not considered. For biological samples, in particular in situ biomolecular reactions, particles and/or biomolecules (such as DNA or proteins) might coat the pore. If this were the case, the electroosmotic contribution to particle motion might become important.3. Existing methods often evaluate the average velocities and electric fields only at the end of the sensing zone.4. Ignoring electro-osmosis greatly limits the choice of nanopore materials that can be used. Many nanopore materials are more highly charged than typical biological particles.5. Accurate measurement of the charge of a particle depends largely on the sampling frequency of the electronics.
Embodiments of the invention overcome or at least mitigate at least some of the problems associated with existing methods by providing a method for the measurement of particle surface charge that: enables particle by particle charge measurements; is able to measure a large number of particles; is not limited by requirements of measuring the system geometry at a high precision prior to analysis; includes a consideration of convection and electroosmosis in its theoretical approach; and incorporates a differential pressure across the nanopore that can be used to slow down the translocation of particles, allowing better resolution of the blockade shape for each particle, which is particularly useful for accurate calibration of the nanopore with particles of a known Zeta Potential.
In certain embodiments, the present invention provides a method of using the size and shape of a signal generated by individual objects travelling through a resistive pulse sensor of either known or unknown geometry, for example a nanopore or micropore (or, more generally, an aperture), to measure their relative electrophoretic mobility. The surface charge density and Zeta Potential of each object can be calculated by calibrating the resistive pulse sensor using particles of defined surface charge density or Zeta Potential respectively.
The objects passing through the pore for analysis can consist of any material including solids (e.g. carbon, silica, polymers, metals), biological particles (e.g. viruses, bacteria, microvesicles, exosomes, liposomes, cells), liquids (e.g. emulsions) or gases (e.g. nanobubbles). In the preferred form solid calibration particles (e.g. carboxylated polystyrene) are used. Objects passing through the pore are therefore referred to as “particles” below.
When each particle passes through the pore, there is a resultant resistive pulse or “blockade”. For objects with a small aspect ratio (largely spherical), the general shape of any given blockade is determined by the shape of the pore this general shape is “stretched” in magnitude (height) and duration (width) depending on the size and the velocity of each particle.
By working with relative magnitudes for each blockade, the difference in particle size can be eliminated from the charge analysis calculations. When the proportional blockade magnitude is equal for any given particles, those particles are at the same position in the pore (or at least are assumed to be at the same position). The relative velocity of those particles can therefore be directly derived by comparing the time it has taken for the particles to get to that point from any consistent defined point on the blockade pulse.
Particles travelling through a pore with a net pressure and voltage bias will have three velocity components:                1. Convection—the pressure driven flow of fluid will carry particles with it        2. Electrophoresis—the particle charge will cause the particles to move through the surrounding fluid towards the oppositely charged electrode        3. Electro-osmosis—the surface charge of the pore membrane (typically negative) attracts a higher density of oppositely charged (typically positive) ions to be present in the vicinity of the pore. These positive ions move towards the negative electrode and carry water molecules with them to create a “plug flow” of fluid.        
Typically, only the electrophoresis component will vary between particles on the basis of surface charge. The present invention provides a method of resolving particle velocity into these three components and deriving the surface charge of an unknown particle by comparing the measured electrophoretic velocity with that of a calibration particle with known surface charge.
For any two particles under the same experimental conditions, the effective particle position within the sensor will be the same when the relative blockade magnitude is the same. Thus for a resistive pulse generated by a particle of unknown surface charge (zeta potential), the measured velocity, along with the known convective and electro-osmotic components derived from calibration particles, are used to calculate electrophoretic velocity of the measured particle. The particle surface charge (zeta potential) is then calculated by comparison of this measured electrophoretic velocity with the calibration particles of known surface charge (zeta potential).
The strength of this novel method is in its simplicity. The method does not require any prior knowledge of the geometry of the sensor, which is prohibitively expensive and time-consuming to measure on a commercial basis (using electron microscopy or similar). The relative electrophoretic velocity of each individual unknown object is simply calibrated against a known particle set based purely on resistive pulse magnitude and shape, with minimal time and processing power required.
This method of charge measurement works best if:                The objects to be measured are suspended in an electrolyte, at a concentration that allows multiple objects to be measured (ideally over a period of a few minutes for operator convenience).        The objects to be measured are smaller than the sensing area to avoid blockages.        Sampling frequency of the current is sufficiently fast to capture a number of data points on each pulse. If sampling frequency is too slow for a particular setup, pulses can be slowed down by application of a pressure differential across the pore or adjustment (even reversal) of the applied bias voltage.        The signal to noise ratio of the pulse is sufficiently large that the reference points of interest can be measured with a good degree of accuracy. If the signal to noise ratio is too small, the signal can be increased by increasing the applied bias voltage and (for flexible pores) reducing the size of the sensor.        Electrophoresis is a significant proportion of the total forces acting on the objects being measured (in particular the calibration particles). This can normally be achieved by increasing bias voltage and reducing pressure differential.        
For measurement of real-life samples with a pore, particularly biologically derived samples, there can be a number of complicating factors. These include:                i) Binding of sample particles (or other components of the sample fluid) to the pore based on opposite polarities.        ii) Non-specific binding of sample particles (or other components of the sample fluid) to the pore.        
Either of these scenarios is likely to cause a change to the Zeta Potential of the membrane ξm, which will be expected to directly affect the calculated value of absolute Zeta Potential for each sample particle.
In cases where the pore is coated by charged particles or biomolecules (such as proteins or DNA) the pore zeta potential can change and affect electroosmosis. The pore zeta potential is evaluated in order to accurately measure particle zeta-potentials. Pore zeta potentials can be determined in situ via streaming potential measurements using variable external pressure applied to the pore. The movement of a liquid through a pore, by applying some variable external pressure creates a streaming potential and streaming current. The pore zetapotential is calculated from the streaming potential vs pressure slope, taking into account the geometry of the pore.
This in situ measurement of the pore zeta potential is useful when chemical and/or biological reactions are monitored in resistive pulse sensors. Different biological systems can affect the pore in very different ways, and checking the Zeta potential of the pore both before and after measuring a biological sample allows these changes to be detected and quantified.
Another approach to compensate for this binding effect is through the addition of calibration particles (of known Zeta Potential) to the sample fluid, which will then be measured under identical conditions to the sample. This method can provide an absolute surface charge measurement even if the Zeta Potential of the pore has been modified by, for example, exosomes, liposomes, proteins, aptamers, DNA, RNA or any other agents. An advantage of this approach is that if the pore characteristics change during, and/or as a result of, the analysis the calibration particles can be used to account for the change through the course of the analysis and provide correct sample measurements. This method can be enhanced through the addition of multiple calibration particle sets each of different known Zeta potentials.
A further approach to treating problems associated with the binding effects is to prevent binding through surface treatment of the pore. This can consist of permanently coating the pore surface with an anti-fouling agent. Alternatively the treatment can involve adding a “blocking agent” to the pore just prior to measurements, which binds to all of the available attachment sites on the surface of the pore and gives a known stable Zeta Potential for the duration of the calibration and sample measurements. An advantage of using a blocking agent is that the blocking agent can be matched to accommodate different sample particle types and carrier fluids.
Aspects of the present invention are set out in the claims.